Orthodox and unorthodox phylogenetic relationships among tunas revealed by the nucleotide sequence analysis of the mitochondrial DNA control region

A phylogeny of all eight recognized taxa of the genus Thunnus was constructed from approximately 400 base pairs of sequence of the mitochondrial DNA (mtDNA) control region. The PCR-amplified control region I segment studied contained a total of 186 variable sites and 159 phylogenetically informative sites. Diagnostic sequences for every taxon were identified. Neighbour-joining phylogenies supported monophyletic origins of the temperate subgenus Thunnus and of the tropical subgenus Neothunnus . Similar results were obtained by maximum parsimony analyses except that there was no support for a monophyletic origin of the subgenus Thunnus . Bigeye tuna, which have been difficult to place in either subgenus using conventional morphological data, was identified as the sister species of Neothunnus . Within the subgenus Thunnus , the Atlantic bluefin and Southern bluefin tunas were shown to be sister taxa of the highly divergent monophyletic clade formed by the Pacific northern bluefin and the Albacore tunas. The conspecific Atlantic ( T. thynnus thynnus ) and Pacific ( T. t. orientalis ) northern bluefin tunas were more divergent (Tamura-Nei distance 0·145 ± 0·019) from each other than the average distance separating most species-pairs within the genus. Thus, a re-examination of their status as subspecies of T. thunnus is warranted.     

Phylogenetic relationships among Thunnus species inferred from rDNA ITS1 sequence

Intra and interspecific nucleotide sequence variation of rDNA first internal transcribed spacer (ITS1) was analysed using all eight species of the genus Thunnus plus two out‐group species within the same family, skipjack tuna Katsuwonus pelamis and striped bonito Sarda orientalis . Intraspecific nucleotide sequence variation in ITS1, including intra‐genomic variation, was low, ranging from 0·003 to 0·014 [Kimura's two parameter distance (K2P)], whereas variation between species within the genus Thunnus ranged from 0·009 to 0·05. The Atlantic and Pacific northern bluefin tunas Thunnus thynnus thynnus and Thunnus thynnus orientalis , recently proposed to be distinct species, were found to share nearly identical ITS1 sequences (mean K2P = 0·006) well within the range of intraspecific variation. The northern bluefin tuna appeared to be a sister group to albacore Thunnus alalunga , with all other Thunnus species in a distinct clade. The ITS1 phylogeny was consistent with mtDNA phylogeny in clustering the three tropical Thunnus species ( T. albacares , T. atlanticus and T. tonggol ). Southern bluefin Thunnus maccoyii and bigeye Thunnus obesus tunas showed a closer affinity to this tropical tuna group than to the northern bluefin tuna and albacore. The molecular data supported mitochondrial introgression between species and contradicted morphological subdivision of the genus into two subgenera Neothunnus and Thunnus .     

Genetics in fisheries management

Genetic analyses have much to offer fisheries managers, especially in the provision of tools enabling unequivocal specimen identification and assessment of stock structure. The three commonly-used genetic tools – allozymes, mitochondrial DNA and microsatellites – differ in their properties. These differences must be born in mind, especially when interpreting gene frequency data collected for stock structure research. Examples where genetic approaches have been used to identify specimens are given, with special attention being given to compliance and labelling issues. Treatment of stock structure focuses on Atlantic cod and on yellowfin, bigeye and albacore tunas. The different resolving powers of the various techniques are discussed. Marine fish typically show low levels of population genetic differentiation, and in such species the use of large sample sizes and, preferably, multiple types of markers are desirable to resolve stock structure issues.     

Habitat and behaviour of yellowfin tuna Thunnus albacares in the Gulf of Mexico determined using pop-up satellite archival tags

This study presents the first data on movement, habitat use and behaviour for yellowfin tuna Thunnus albacares in the Atlantic Basin. Six individuals were tracked in the Gulf of Mexico using pop-up satellite archival tags. Records up to 80 days in length were obtained, providing information on depth and temperature preferences as well as horizontal movements. Thunnus albacares in the Gulf of Mexico showed a strong preference for the mixed layer and thermocline, consistent with findings for this species in other ocean basins. Fish showed a diel pattern in depth distribution, remaining in surface and mixed layer waters at night and diving to deeper waters during the day. The vertical extent of T. albacares habitat appeared to be temperature limited, with fish generally avoiding waters that were >6° C cooler than surface waters. The vertical and thermal habitat usage of T. albacares differs from that of bigeye Thunnus obesus and bluefin Thunnus thynnus , Thunnus orientalis and Thunnus maccoyii tunas. These results are consistent with the results of earlier studies conducted on T. albacares in other oceans.     

Phylogenetic relationships between tuna species of the genus Thunnus (Scombridae: Teleostei): Inconsistent implications from morphology,nuclear and mitochondrial genomes

In order to infer phylogenetic relationships between tuna species of the genus Thunnus, partial sequences of the mitochondrial cytochrome b and ATPase genes were determined in all eight species. Supplemental restriction analysis on the nuclear rRNA gene was also carried out. Pacific northern bluefin tuna (Thunnus thynnus orientalis) was found to have mtDNA distinct from that of the Atlantic subspecies (T. t. thynnus) but very similar to that from the species albacore (T. alaluga). In contrast, no differentiation in nuclear genome was observed between the Atlantic and Pacific northern bluefin tunas. The Atlantic northern bluefin and southern bluefin tunas possessed mtDNA sequences very similar to species of yellowfin tuna group and not so similar to albacore and bigeye tunas which were morphologically assigned to the bluefin tuna group. The molecular data indicate that (1) mtDNA from albacore has been incorporated into the Pacific population of northern bluefin tuna and has extensively displaced the original mtDNA, and (2) albacore is the earliest offshoot, followed by bigeye tuna in this genus, which is inconsistent with the phylogenetic relationships between these tuna species inferred from morphology. Correspondence to: S. Chow     

Microsatellite DNA markers for population‐genetic studies of Atlantic bluefin tuna (Thunnus thynnus thynnus) and other species of genus Thunnus

Twenty‐five microsatellites from Atlantic bluefin tuna (Thunnus thynnus thynnus) were characterized. All 25 microsatellites were polymorphic; the number of alleles among up to 56 individuals surveyed ranged from two to 23. Atlantic bluefin tuna are highly exploited and major questions remain as to stock structure and abundance in the eastern and western North Atlantic. The microsatellites will be useful in testing stock‐structure hypotheses and in generating estimates of effective population size. The polymerase chain reaction primer sets developed also amplified identifiable alleles in three other species of genus Thunnus: T. albacares (yellowfin tuna), T. alalunga (albacore tuna) and T. obesus (bigeye tuna).     

The genetic population structure of Thunnus thynnus (Linnaeus, 1758) in the Mediterranean Sea,a controversial issue

The Atlantic bluefin tuna (ABFT), Thunnus thynnus (Linnaeus, 1758), is an important commercial species managed as two different stocks, western and eastern Atlantic, with their spawning grounds in the Gulf of Mexico and in the Mediterranean Sea, respectively. The eastern Atlantic stock has been overexploited in the last decades, leading to the application of specific management measures introduced by the International Commission for the Atlantic Tuna (ICCAT). A clear understanding of the genetic structure of ABFT Mediterranean population should be pursued in order to support management decisions. To date the genetic studies on the Mediterranean ABFT, carried out with different molecular markers and sampling procedures, have produced unclear results. Here, we analysed ABFT samples from central and western Mediterranean Sea with mitochondrial sequences and 11 microsatellite loci to investigate, among the others, the area of the Strait of Messina, where environmental conditions seem to support a resident population of ABFT. Furthermore, genetic analyses of mitochondrial sequences were carried out including nucleotide sequences of Adriatic ABFT wild larvae retrieved from GenBank. Among the investigated areas a genetic differentiation was detected between the Strait of Messina and the Tyrrhenian Sea with microsatellite loci according to the exact G test, but not to the Bayesian analyses carried out with STRUCTURE. The analyses with mitochondrial sequences do not reveal any differentiation among sampled areas, however, a highly significant genetic divergence was observed between the Adriatic mitochondrial sequences retrieved from GenBank and the central‐western Mediterranean sequences obtained in the present work. Our results provide some evidence of population structure of Mediterranean ABFT adding pieces to a still unclear picture.     

Spawning of bluefin tuna in the black sea: historical evidence, environmental constraints and population plasticity

The lucrative and highly migratory Atlantic bluefin tuna, Thunnus thynnus (Linnaeus 1758; Scombridae), used to be distributed widely throughout the north Atlantic Ocean, Mediterranean Sea and Black Sea. Its migrations have supported sustainable fisheries and impacted local cultures since antiquity, but its biogeographic range has contracted since the 1950s. Most recently, the species disappeared from the Black Sea in the late 1980s and has not yet recovered. Reasons for the Black Sea disappearance, and the species-wide range contraction, are unclear. However bluefin tuna formerly foraged and possibly spawned in the Black Sea. Loss of a locally-reproducing population would represent a decline in population richness, and an increase in species vulnerability to perturbations such as exploitation and environmental change. Here we identify the main genetic and phenotypic adaptations that the population must have (had) in order to reproduce successfully in the specific hydrographic (estuarine) conditions of the Black Sea. By comparing hydrographic conditions in spawning areas of the three species of bluefin tunas, and applying a mechanistic model of egg buoyancy and sinking rate, we show that reproduction in the Black Sea must have required specific adaptations of egg buoyancy, fertilisation and development for reproductive success. Such adaptations by local populations of marine fish species spawning in estuarine areas are common as is evident from a meta-analysis of egg buoyancy data from 16 species of fish. We conclude that these adaptations would have been necessary for successful local reproduction by bluefin tuna in the Black Sea, and that a locally-adapted reproducing population may have disappeared. Recovery of bluefin tuna in the Black Sea, either for spawning or foraging, will occur fastest if any remaining locally adapted individuals are allowed to survive, and by conservation and recovery of depleted Mediterranean populations which could through time re-establish local Black Sea spawning and foraging.     

Distinct Yellowfin Tuna (Thunnus albacares) Stocks Detected in Western and Central Pacific Ocean (WCPO) Using DNA Microsatellites

The yellowfin tuna, Thunnus albacares (Bonnaterre, 1788), covers majority of the Philippines’ tuna catch, one of the major fisheries commodities in the country. Due to its high economic importance sustainable management of these tunas has become an imperative measure to prevent stock depletion. Currently, the Philippine yellowfin tuna is believed to be part of a single stock of the greater WCPO though some reports suggest otherwise. This study therefore aims to establish the genetic stock structure of the said species in the Philippines as compared to Bismarck Sea, Papua New Guinea using nine (9) DNA microsatellite markers.DNA microsatellite data revealed significant genetic differentiation between the Philippine and Bismarck Sea, Papua New Guinea yellowfin tuna samples. (FST = 0.034, P = 0.016), which is further supported by multilocus distance matrix testing (PCoA) and model-based clustering (STRUCTURE 2.2).With these findings, this study posits that the yellowfin tuna population in the Philippines is a separate stock from the Bismarck Sea population. These findings add evidence to the alternative hypothesis of having at least 2 subpopulations of yellowfin tuna in the WCPO and calls for additional scientific studies using other parameters to investigate this. Accurate population information is necessary in formulating a more appropriate management strategy for the sustainability of the yellowfin tuna not only in the Philippines but also in the WCPO.     

New data on Oncophora melanocephala (Nematoda: Camallanidae), a little-known parasite of scombrid fishes

The camallanid nematode Oncophora melanocephala (Rudolphi, 1819) is redescribed from specimens collected in tunas, Thunnus thynnus (L.), T. albacares (Bonnaterre), T. atlanticus (Lesson) and Auxis thazard (Lacépède) from the Atlantic littoral of Rio de Janeiro, Brazil. In addition to a specific structure of the buccal capsule, the species is characterized by the presence of seven pairs of preanal and five pairs of postanal subventral papillae in the male and by an unusual shape of body in the adult female; deirids are described for the first time in O. melanocephala. The structure of the buccal capsule of O. melanocephala, particularly the presence of a large posterior portion of capsule and a colourless connecting ring between buccal capsule and oesophagus, shows close affinities of Oncophora Diesing, 1851 with Paracamallanus Yorke et Maplestone, 1928. The findings of O. melanocephala in T. atlanticus and A thazard represent new host records.     

Discrimination of juvenile yellowfin (Thunnus albacares) and bigeye (T. obesus) Tunas using mitochondrial DNA control region and liver morphology

Yellowfin tuna, Thunnus albacares (Bonnaterre, 1788) and bigeye tuna, Thunnus obesus (Lowe, 1839) are two of the most economically important tuna species in the world. However, identification of their juveniles, especially at sizes less than 40 cm, is very difficult, often leading to misidentification and miscalculation of their catch estimates. Here, we applied the mitochondrial DNA control region D-loop, a recently validated genetic marker used for identifying tuna species (Genus Thunnus), to discriminate juvenile tunas caught by purse seine and ringnet sets around fish aggregating devices (FADs) off the Southern Iloilo Peninsula in Central Philippines. We checked individual identifications using the Neighbor-Joining Method and compared results with morphometric analyses and the liver phenotype. We tested 48 specimens ranging from 13 to 31 cm fork length. Morpho-meristic analyses suggested that 12 specimens (25%) were bigeye tuna and 36 specimens (75%) were yellowfin tuna. In contrast, the genetic and liver analyses both showed that 5 specimens (10%) were bigeye tuna and 43 (90%) yellowfin tuna. This suggests that misidentification can occur even with highly stringent morpho-meristic characters and that the mtDNA control region and liver phenotype are excellent markers to discriminate juveniles of yellowfin and bigeye tunas.     

Differentiation of five tuna species by a multiplex primer-extension assay

A novel methodology based on analysis of mtDNA-cytb diagnostic sites was performed to discriminate four closely related species of Thunnus (Thunnus alalunga, Thunnus albacares, Thunnus obesus and Thunnus thynnus) and one species of Euthynnus (Katsuwonus pelamis) genus in raw and canned tuna. The primers used in the preliminary PCR designed in well conserved region upstream and downstream of the diagnosis sites successfully amplified a 132bp region from the cytb gene of all the species taken into consideration. The sites of diagnosis have been interrogate simultaneously using a multiplex primer-extension assay (PER) and the results were confirmed by fragment sequencing. The applicability of the multiplex PER assay to commercial canned tuna samples was also demonstrated. The proposed test could be useful for detection of fraud and for seafood traceability.     

Microsatellite and mitochondrial DNA analyses of Atlantic bluefin tuna (Thunnus thynnus thynnus) population structure in the Mediterranean Sea

Genetic variation was surveyed at nine microsatellite loci and the mitochondrial control region (868 bp) to test for the presence of genetic stock structure in young-of-the-year Atlantic bluefin tuna (Thunnus thynnus thynnus) from the Mediterranean Sea. Bluefin tuna were sampled over a period of 5 years from the Balearic and Tyrrhenian seas in the western basin of the Mediterranean Sea, and from the southern Ionian Sea in the eastern basin of the Mediterranean Sea. Analyses of multilocus microsatellite genotypes and mitochondrial control region sequences revealed no significant heterogeneity among collections taken from the same location in different years; however, significant spatial genetic heterogeneity was observed across all samples for both microsatellite markers and mitochondrial control region sequences (FST=0.0023, P=0.038 and PhiST=0.0233, P=0.000, respectively). Significant genetic differentiation between the Tyrrhenian and Ionian collections was found for both microsatellite and mitochondrial markers (FST=0.0087, P=0.015 and PhiST=0.0367, P=0.030, respectively). These results suggest the possibility of a genetically discrete population in the eastern basin of the Mediterranean Sea.     

Genetic diversity and historical demography of Atlantic bigeye tuna (Thunnus obesus)

Bigeye (Thunnus obesus) is a large, pelagic, and migratory species of tuna that inhabits tropical and temperate marine waters worldwide. Previous studies based on mitochondrial RFLP data have shown that bigeye tunas from the Atlantic Ocean are the most interesting from a genetic point of view. Two highly divergent mitochondrial haplotype clades (I and II) coexist in the Atlantic Ocean. One is almost exclusive of the Atlantic Ocean whereas the other is also found in the Indo-Pacific Ocean. Bigeye tuna from the Atlantic Ocean is currently managed as a single stock, although this assumption remains untested at the genetic level. Therefore, genetic diversity was determined at the mitochondrial control region to test the null hypothesis of no population structure in bigeye tuna from the Atlantic Ocean. A total of 331 specimens were sampled from four locations in the Atlantic Ocean (Canada, Azores, Canary Islands, and Gulf of Guinea), and one in the Indian and Pacific Oceans, respectively. The reconstructed neighbor-joining phylogeny confirmed the presence of Clades I and II throughout the Atlantic Ocean. No apparent latitudinal gradient of the proportions of both clades in the different collection sites was observed. Hierarchical AMOVA tests and pairwise phi(ST) comparisons involving Atlantic Ocean Clades I and II were consistent with a single stock of bigeye tuna in the Atlantic Ocean. Population genetic analyses considering phylogroups independently supported gene flow within Clade II throughout the Atlantic Ocean, and within Clade I between Atlantic and Indo-Pacific Oceans. The latter result suggests present uni-directional gene flow from the Indo-Pacific into the Atlantic Ocean. Moreover, mismatch analyses dated divergence of Clades I and II during the Pleistocene, as previously proposed. In addition, migration rates were estimated using coalescent methods, and showed a net migration from Atlantic Ocean feeding grounds towards the Gulf of Guinea, the best-known spawning ground of Atlantic bigeye tuna.     

Characterization of two tropomyosin isoforms from the fast skeletal muscle of bluefin tuna Thunnus thynnusorientalis

Fast skeletal muscle tropomyosin (TM) of tunas is composed of nearly equimolar amount of two isoforms designated α-TM and β-TM expediently based on their migration behavior in SDS-PAGE, whereas corresponding TMs from the other fish species are homogenous (α-type). The presence of β-TM is thus specific to tunas so far. The amino acid sequence of β-TM from bluefin tuna Thunnus thynnus orientalis, which has not been revealed to date unlike α-TM, was successfully obtained in this study by cDNA cloning. The coding region of β-TM cDNA comprised of an open reading frame of 855 bp encoding 284 amino acid residues, like most of the TMs. Unexpectedly, the sequence of β-TM showed high similarity to those of other vertebrate α-type TMs including tuna α-TM. Phylogenetic analysis also showed that β-TM has the closest relationship with α-TM of tuna. This fact was quite unlike the relation of mammalian α- and β-TMs. Based on the distribution of amino acid substitutions, it was suggested that tuna TM isoforms are the products of different genes. By thermodynamic analysis of native and reconstituted TMs, it was demonstrated that β-TM is less thermostable than α-TM. Proteolytic digestion also supported the lower stability of the former.     

Single nucleotide polymorphism discovery in albacore and Atlantic bluefin tuna provides insights into worldwide population structure

The optimal management of the commercially important, but mostly over‐exploited, pelagic tunas, albacore (Thunnus alalunga Bonn., 1788) and Atlantic bluefin tuna (BFT; Thunnus thynnus L., 1758), requires a better understanding of population structure than has been provided by previous molecular methods. Despite numerous studies of both species, their population structures remain controversial. This study reports the development of single nucleotide polymorphisms (SNPs) in albacore and BFT and the application of these SNPs to survey genetic variability across the geographic ranges of these tunas. A total of 616 SNPs were discovered in 35 albacore tuna by comparing sequences of 54 nuclear DNA fragments. A panel of 53 SNPs yielded F_ST values ranging from 0.0 to 0.050 between samples after genotyping 460 albacore collected throughout the distribution of this species. No significant heterogeneity was detected within oceans, but between‐ocean comparisons (Atlantic, Pacific and Indian oceans along with Mediterranean Sea) were significant. Additionally, a 17‐SNP panel was developed in Atlantic BFT by cross‐species amplification in 107 fish. This limited number of SNPs discriminated between samples from the two major spawning areas of Atlantic BFT (F_ST = 0.116). The SNP markers developed in this study can be used to genotype large numbers of fish without the need for standardizing alleles among laboratories.     

Permanent genetic resources added to Molecular Ecology Resources Database 1 October 2010-30 November 2010

This article documents the addition of 277 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Ascochyta rabiei, Cambarellus chapalanus, Chionodraco hamatus, Coptis omeiensis, Cynoscion nebulosus, Daphnia magna, Gerbillus nigeriae, Isurus oxyrinchus, Lates calcarifer, Metacarcinus magister, Oplegnathus fasciatus, Pachycondyla verenae, Phaethon lepturus, Pimelodus grosskopfii, Rotylenchulus reniformis, Scomberomorus niphonius, Sepia esculenta, Terapon jarbua, Teratosphaeria cryptica and Thunnus obesus. These loci were cross-tested on the following species: Austropotamobius italicus, Cambarellus montezumae, Cambarellus puer, Cambarellus shufeldtii, Cambarellus texanus, Chionodraco myersi, Chionodraco rastrospinosus, Coptis chinensis, Coptis chinensis var. brevisepala, Coptis deltoidea, Coptis teeta, Orconectes virilis, Pacifastacus leniusculus, Pimelodus bochii, Procambarus clarkii, Pseudopimelodus bufonius, Rhamdia quelen, Sepia andreana, Sepiella maindroni, Thunnus alalunga, Thunnus albacares, Thunnus maccoyii, Thunnus orientalis, Thunnus thynnus and Thunnus tonggol.     

Longtail tuna,Thunnus tonggol (Bleeker, 1851): a global review of population dynamics,ecology, fisheries,and considerations for future conservation and management

Longtail tuna (Thunnus tonggol) is a neritic species that supports commercial, artisanal and recreational fisheries throughout the Indo-Pacific region. Historically receiving little attention by commercial fisheries, the global annual catch of longtail tuna has steadily risen from around 30,000 t in the early 1980s to exceeding 200,000 t since 2004, reaching a peak of 291,264 t in 2007, and was 281,613 t in 2017. Catches of longtail tuna in the Indian Ocean now exceed catches of principal commercial target species, such as albacore and bigeye tunas. A sequence of stock assessments undertaken throughout the species’ range since the late 1980s persistently indicated that at least three of the four stocks defined in this paper are likely to have been, and most likely are currently, subject to overfishing and overfished as a result of excess fishing effort on this relatively slow-growing and long-lived tuna species. As the spawning biomass of principal tuna target species continue to decline in both the Indian and western and central Pacific Oceans, the increasing catches of longtail tuna, other neritic tunas, and seerfishes is worrisome. Few conservation and management measures (CMMs) are currently in place specifically for longtail tuna, although in recent years some coastal States, Regional Fishery Bodies, and tuna Regional Fisheries Management Organisations have begun to develop initiatives to improve the catch and biological data quality for longtail tuna and sympatric species of neritic tunas and tuna-like species. This paper provides a global review of biological, ecological and fishery information to provide researchers, fishery managers and policy makers with the most current information from which to begin to guide future stock assessment and the development of CMMs for longtail tuna.

     

Longtail tuna Thunnus tonggol (Bleeker, 1851) shows genetic partitioning across,but not within,basins of the Indo‐Pacific based on mitochondrial DNA

Genetic stock structure is atypical in tuna species, with most species demonstrating geographically‐broad, panmictic populations. Here, genetic data suggest a distinct pattern for Thunnus tonggol across the Indo‐Pacific region. The genetic variation in the coastal tuna T. tonggol sampled from across the South China Sea was examined using the highly variable mitochondrial DNA displacement loop (D‐loop) gene region. One hundred and thirty‐nine specimens were sampled from four locations in Indonesia, Vietnam and the Philippines. Phylogenetic reconstruction of genetic relationships revealed no significant ?_ST statistics and hence no population structure within the South China Sea. However, subsequent analysis with sequence data from coastal northwest India infers discrete genetic stocks between the Indian Ocean and the South China Sea. Consistent with previous genetic analyses of tuna species in the Indo‐Pacific, the findings in this study infer no population structure within each basin, but rather show a significant partitioning across the wider region. Furthermore, these results have implications for the management of the commercially valuable Thunnus tonggol across national boundaries, and thus requiring collaboration among countries to ensure its sustainable use.     

Characterization of microsatellite loci from the Japanese eel Anguilla japonica

The Japenese eel, Anguilla japonica, is generally assumed to be composed of a single population with wide distribution range, and some genetic studies using allozyme or mitochondrial DNA methods supported this population model. However, one genetic study suggested the existence of multiple populations in this species, and thus, more detailed studies on the population structure is needed. Here we characterized a total of 11 microsatellite markers of the Japanese eel. These will serve as powerful tools for detailed population study for the Japanese eel, though two of them showed the significant departure from the Hardy–Weinberg expectations.